This application claims priority to the foreign application KR 2001-43450 filed Jul. 19, 2001.
The present invention relates to a method for identifying Mycobacterium tuberculosis (hereinafter, referred to as xe2x80x98M. tuberculosisxe2x80x99) and Mycobacterium Other Than Tuberculosis (hereinafter, referred to as xe2x80x98MOTTxe2x80x99), and for the determination of resistance of M. tuberculosis to an antituberculosis drug obtained by the mutation of the rpoB gene.
Tuberculosis is a chronic wasting disease caused by M. tuberculosis. Worldwide, it ranks the first in mortality and morbidity among infectious diseases (Global Tuberculosis Programme. Global Tuberculosis Control, WHO Report 1997. World Health Organization, 1997). Carriers of M. tuberculosis presently number about 1.9 billion, a third of the world population, and about 8-10 million of these carriers develop into new tuberculosis patients per year, and about 3 million of patients die of tuberculosis per year (Dolin P. J., Raviglion M. C., Kochi, A. (1994) Global tuberculosis incidence and mortality during 1990-2000. Bull World Health Organization. 72(2): 213-220; Kochi, A. (1992) The global tuberculosis situation and the new control strategy of the World Health Organization. Tubercle. SCI. 72:1-6; Styblo K. Epidemiology of Tuberculosis the Hague, Royal Netherland Tuberculosis Association (1991), p. 83 in: Minister of Health and Welfare, The Korean National Tuberculosis Association. Measures for Tuberculosis Control in 2000s, p5, 1997). Also, about a half of the population in Korea are carriers of M. tuberculosis, and about 150,000 persons develop into new tuberculosis patients per year, and about 14,000 patients per year die of tuberculosis (Sreevatsan S. Stockbauer K E, Pan X, Kreiswirth B N, Mogha S L, Jacobs W R Jr, Telenti A, Musser J M. (1997) Ethambutol resistance in M. tuberculosis: critical role of embB mutations. Antimicrob Agents Chemother 41:1677-1681).
Recently, it was reported that patients doubly infected with HIV virus and M. tuberculosis are much more susceptible to tuberculosis. The seriousness of the problem of tuberculosis has become increasingly apparent, together with the increase of HIV in the world as well. Presently, it is estimated that about 15 million patients are double infected by both the M. tuberculosis and HIV. Also, most of these patients will probably develop into fulminant tuberculosis patients, which will lead to their deaths (Narain J. P., Raviglione M. C., Kochi A. (1992) HIV-associated tuberculosis in developing countries: epidemiology and strategies for prevention. Tuber Lung Dis. SCI. 73(6): 311-321).
The insufficiency of the present antituberculosis drugs and improper treatment and control of tuberculosis in these developing countries results in the increase of patients with M. tuberculosis which are the resistant to antituberculosis drugs (Global Tuberculosis Programme. Anti-tuberculosis drug resistance, WHO Report 1997. World Health Organization, 1997). The increasing appearance of resistant M. tuberculosis has resulted in the concomitant increase in tuberculosis related mortality and has obstructed attempts to eliminate it from a population (Ariel, P. M., M. C. Raiglion, A. Laszlo, N. Binkin, H. L. Rieder, F. Buster, D. L. Cohn, C. S. B. L. van weezenbeck, S. J. Kim, P. Chaulet, P. Nunn. (1998) Global surveillance for antituberculosis-drug resistance. New England J. of Medicine. SCI. 338:1641-1649; Centers for Disease Control. (1992) National action plan to combat multidrug-resistant tuberculosis. Morb Mortal Wkly Rep 41(RR-11):5-48; Global Tuberculosis Programme. Global project on Anti-tuberculosis Drug Resistance Surveillance. WHO Report 1997. World Health Organization, 1997; Tuberculosis: A Global Emergency (news). World Health Forum, 14(4):438, 1993. In: Minister of Health and Welfare, The Korean National Tuberculosis Association. Measures for Tuberculosis Control in 2000s, 1997). In the meantime, current treatment of resistant M. tuberculosis is quite costly and has low efficiency, thus the resistant mycobacteria often develops into incurable tuberculosis. Therefore, earlier diagnosis of resistant M. tuberculosis should result in more efficient treatment of tuberculosis patients.
In most countries, including Korea, the drug-susceptibility test for M. tuberculosis is performed using standard microbiological methods, which require long time periods of 8-10 weeks to grow M. tuberculosis in culture (Index of Korean Health and Welfare in 1996, p. 412, Korea Institute for Health and Social Affair, 1997). Therefore, methods for rapidly and accurately detecting the drug susceptibility of the infecting M. tuberculosis are needed. Rapid diagnosis of resistant mycobacteria may increase the efficiency of the tuberculosis treatment because it may provide proper treatment strategies earlier in the development of the disease. Ultimately, rapid diagnostic methods may slow the increase of incurable tuberculosis resulting from infection with drug-resistant M. tuberculosis. Therefore, such diagnostic methods are important health care technologies capable of decreasing further economic loss by prevention, elimination and treatment of tuberculosis.
Recently, the mechanism of drug-resistance was discovered by the use of genetic-based technology. For example, it is reported that the mechanism by which M. tuberculosis obtains resistance to rifampin, one of the strongest effective antituberculosis drugs, results from a nucleotide mutation in the 69bp region of the gene which encodes the xcex2-subunit of RNA polymerase (rpoB gene) (Telenti A., Imboden P., Marchesi F., Lowrie D., Cole S., Colston M. J., Matter L., Schopfer K., Bodmer T. (1993) Detection of rifampicin-resistance mutation in Mycobacterium tuberculosis. Lancet. SCI. 341:647-50; Vareldzis B. P., Grosset J., de kantor I., Crofton J., Laszlo A., Felten M., Raviglione M. C., Kochi A. (1994) Drug-resistant Tuberculosis: Laboratory Issues, World Health Organization Recommendations. Tuber Lung Dis. SCI. 75(1):1-7). At least 97% of M. tuberculosis with resistance to rifampin is due to the above mutation.
The genus Mycobacterium includes, in addition to M. tuberculosis, M. lepraeae (Hansen""s disease or Leprosy) and other Mycobacterium species generically called Mycobacterium Other Than Tuberculosis (hereinafter, referred to as xe2x80x98MOTTxe2x80x99). MOTTs are mycobacteria which cause opportunistic infections, and thus MOTTs generally infect patients who have reduced immunity; however, normal persons can be infected on occasion. Since 1980, most of the industrialized nations have reported that MOTTs cause tuberculosis in HIV patients. Further, the number of diseases found to be caused by MOTTs has been increasing, so that the rapid and accurate identification of mycobacterial species has been recognized as being important for health care providers.
Conventional methods for identifying M. tuberculosis and MOTTs are based on various microbiological and biochemical properties of Mycobacterium species (Kochi A., Vareldzis B., Styblo K. (1993) Multidrug-resistant tuberculosis and its control. Res Microbiol. SCI. 144(2):104-110). However, such conventional identifications require for up to four weeks of growth time, depending on the types of mycobacteria, thus the identification time is prolonged. In addition, the results obtained by the conventional methods are sometimes unclear, and some Mycobacterium species cannot be distinguished from other species with these conventional methods. Therefore, to overcome the above problems of the conventional identifications, the identification of MOTTs using molecular biological methods has been recently developed.
Particularly, methods for the molecular biological identification of MOTTs using as its target, a highly conserved genetic domain in all mycobacterial species, were developed (Timpe A, Runyon E H: The relationship of xe2x80x9catypicalxe2x80x9d acid-fast bacteria to human disease: A preliminary report. J Lab Clin Med. 44: 202, 1954; Jenkins, P. A.: Lipid analysis for the identification of mycobacteria. An appraisal. Rev. Infect. Dis. 3: 382-866, 1981; Tsang, A., I. Drupa, M. Goldgerg, J. McClatchy, and P. Brennan. 1983. Use of serology and thin-layer chromatography for the assembly of an authenticated collection of serovars within the Mycobacterium avium-Mycobacterium intracellulare-Mycobacterium scrofulaceum complex. Int. J. Syst. Bacteriol.; Butler, W. R., K. C. Jost, Jr., and J. O. Kilburn. 1991. Identification of mycobacteria by high-performance liquid chromatography. J. Clin. Microbiol. 29:2468-2472, 33:285-292).
Among these, the rpoB gene found by the present inventors may be used in identifying MOTTs more simply and rapidly than the conventional molecular biological method using identification of the nucleotide polymorphism of 16S rRNA (KR99-46795).
That is to say, the rpoB gene includes highly conserved regions capable of being detected in all the species of mycobacteria. More importantly, the rpoB genes from Entero-bacteriaceae are not amplified by PCR using the primers based on the rpoB gene sequence of mycobacterial species, or if amplified, the PCR products are distinguishable from each other. Therefore, the mycobacterial rpoB gene may be used in preparation of PCR primers specific to Mycobacterium species.
Further, the results of the above prior studies by the present inventors show that the rpoB gene of Mycobacterium includes nucleotide regions having polymorphisms. Therefore, the rpoB gene may also be used in preparation of Mycobacterium species-specific probes for DNA-hybridization.
Conclusively, the present inventors found in prior studies that a 361bp region in the rpoB gene can be successfully used in the specific identification of M. tuberculosis and MOTTs.
An object of the present invention is to provide a method for specifically identifying M. tuberculosis and MOTTs, and to detect whether said mycobacteria has a mutation of the rpoB gene, rendering it drug resistant.
Another object of the present invention is to provide primers for PCR amplification of the rpoB gene used in identifying M. tuberculosis and MOTTs, and to detect the presence of the mutation of the rpoB gene.
Still another object of the present invention is to provide oligomer probes and a membrane to adhere said probes for performing reverse blot hybridization.
Yet another object of the present invention is to provide a kit for identifying M. tuberculosis and MOTTs, together with detecting the antimicrobial resistance of a mycobacterial species by detection of mutations of the rpoB gene, using oligomer probes adhered to the membrane and said primers, or which includes said primers, said oligomer probes and said membranes.
To achieve the above purposes, the present invention identifies M. tuberculosis and MOTTs, and detects whether the Mycobacterium has a mutation in the rpoB gene which would confer antibiotic resistance by the following method:
(1) isolating DNA from a sample;
(2) amplifying a 531bp fragment of the rpoB gene of the Mycobacterium by PCR using said mycobacterial DNA isolated in step (1) as template, and using the primers MOTT-rpo-long-B-5xe2x80x2 (5xe2x80x2-TCAAGGAGAAGCGCTACGACCTGGC-3xe2x80x2; SEQ. ID. NO. 1) and TR8-long-NB-3xe2x80x2(5xe2x80x2-ACGGGTGCACGTCGCGGACCTCCA-3xe2x80x2; SEQ. ID. NO. 2); and,
(3) performing PCR-reverse blot hybridization by hybridizing the PCR products obtained in step (2) to a membrane capable of adhering the oligomer probes of SEQ. ID. NOs. 3 to 30, wherein the oligomer probes of SEQ. ID. NOs. 3 to 20 are species-specific oligomer probes capable of binding to a specific DNA sequence of a specific mycobacterial species and wherein the oligomer probes of SEQ. ID. NOs. 21 to 30 are capable of hybridizing with specificity to mutants of the rpoB gene which confer drug resistance on M. tuberculosis or which are also capable of hybridizing to the wild type rpoB gene.
The present invention is explained in detail below.
In step (1), the samples are prepared from patients who are carriers or expected carriers of M. tuberculosis. Because the Mycobacterium infects the lungs, samples are preferably obtained from patient expectoration. The isolation of DNA from said samples may be performed by methods commonly known to a person skilled in the art.
In step (2), the primers MOTT-rpo-long-B-5xe2x80x2 (SEQ ID NO. 1) and TR8-long-NB-3xe2x80x2 (SEQ ID NO. 2) are used to amplify a 531bp fragment of the Mycobacterium rpoB gene by PCR, as illustrated in FIG. 1. As shown in FIG. 2, the 531bp PCR product includes i) a conserved sequence native to all Mycobacterium, ii) a species-specific polymorphic sequence, and iii) the sequence which, when mutated, confers drug resistance upon the host M. tuberculosis. Said amplified PCR product of the mycobacterial rpoB gene may be used to identify and distinguish between M. tuberculosis and MOTTs, and to detect a mutation in the rpoB gene. Such a mutation in the rpoB gene renders M. tuberculosis resistant to drugs such as rifampin, rifamycin, or derivatives thereof.
In step (3), the rpoB gene PCR product obtained in step (2) is hybridized with the oligomer probes of Table 1 below using the reverse blot hybridization method (Sambrook and Russell, Molecular Cloning: A laboratory manual, third edition, Cold Spring Harbor Press, New York, 2001). The oligomer probes of Table 1 below include oligomer probes that are capable of hybridizing with a specific DNA sequence of a specific species of Mycobacterium, and that are capable of hybridizing with specificity to the wild type rpoB gene or to mutated DNA sequences of the rpoB gene which confer resistance upon the host M. tuberculosis to drugs such as rifampin.
The oligomer probes described by SEQ ID NOs. 3 to 20 in Table 1 were constructed to hybridize with a specific DNA sequence of a specific species of Mycobacterium.
The oligomer probes WT1, WT2, WT3, WT4 and WT5 (SEQ ID NOs. 21-25) can be used to identify the wild type rpoB gene of M. tuberculosis by hybridizing with nucleotide sequences 509-514, 515-520, 521-529, 525-529 and 530-534, respectively, of the PCR product. The oligomer probes MT1, MT2, MT3, MT4 and MT5 (SEQ ID NOs. 26-30) can be used to detect mutations in the M. tuberculosis rpoB gene, specifically, TTG mutation of nucleotide 531, AAC mutation of nucleotide 526, GTC mutation of nucleotide 516, CCA mutation of nucleotide 516, and CCG mutation of nucleotide 511, respectively.
FIG. 3 shows the absence of bands in the reverse blot hybridization using the oligomer probes WT1-5. The failure of the probes to bind to their DNA recognition sequences indicates the presence of mutations in the region encoding susceptibility to rifampin. Therefore, the detection of said mutant types enables the determination of M. tuberculosis resistance to rifampin.
The oligomer probes in Table 1 were designed to hybridize at the same temperature and be highly specific for a particular species of Mycobacterium. To achieve these purposes, the length, GC content and positions of possible mismatch in the oligomer probes were modulated. For some less sensitive oligomer probes, non-specific nucleotides were added to the 5xe2x80x2-terminal ends in order to increase the efficiency of hybridization with the corresponding target DNA sequence.
Stable adhesion of the oligomer probes to the membrane is a result of a covalent linkage formed between an amino group conjugated to the 5xe2x80x2-terminal end of the oligomer probe and a carboxyl group on the membrane surface. These covalent links prevent the oligomer probes from moving freely on the membrane during the reverse blot hybridization. The present invention employs, but is not limited to, Biodyne-C membrane (Pall Biosupport, East Hills, N.Y.), a negatively charged nylon membrane, as any membranes with carboxyl groups on the surface may be preferably used.
In summary, the present invention provides primers for amplifying a 531bp fragment of the M. tuberculosis rpoB gene by PCR, the oligomer probes described in Table 1 and the membrane for adhesion to said oligomer probes, in order to specifically identify M. tuberculosis and MOTTs, and to detect mutations in the rpoB gene which confer drug resistance to the host Mycobacterium.
Further, the present invention provides a kit for the separate identification of M. tuberculosis and MOTTs, and for the detection of mutations in the rpoB gene which confers drug resisistance to the Mycobacterium, using said primers, the oligomer probes of Table 1 and the membrane for adhering said oligomer probes.